Fragment ion spectra supply information about the structure of the fragmented ions; for peptides they provide considerable information about the sequence of the amino acids. The acquisition of daughter or fragment ion spectra basically requires (a) a station for selecting the ions to be fragmented (the “parent ions”), (b) a station for fragmenting the parent ions, and (c) a station for analyzing the fragment ions. For selecting the parent ions, a filtering mass spectrometer is required; for the analysis, a mass spectrometer which acquires the daughter ion spectrum. Therefore the term “tandem mass spectrometry” is used.
There are two fundamentally different methods for the acquisition of daughter ion spectra in time-of-flight mass spectrometers:
The first method, which, in commercial embodiments, always involves ionization of the analyte molecules by matrix-assisted laser desorption (MALDI), uses so-called TOF/TOF mass spectrometers. TOF/TOF mass spectrometers are two-stage time-of-flight mass spectrometers, in whose first stage the ions are both selected and fragmented, and in whose second stage the fragment ions are measured separately as daughter ion spectra after a further acceleration phase. The ions can be fragmented by laser-induced metastable decomposition (LID, also termed post source decay or PSD) in the flight region of the first stage, or by collision-induced decomposition (CID) in a collision cell at collision energies of around two kiloelectron-volts. These methods and the corresponding instruments are described in DE 198 56 014 C2 (U.S. Pat. No. 6,300,627 B1, C. Köster, A. Holle, J. Franzen) and in U.S. Pat. No. 6,348,688 B1 (M. L. Vestal, S. C. Gabeler), U.S. Pat. No. 6,441,369 B1 (M. L. Vestal, S. C. Gabeler), U.S. Pat. No. 6,534,764 B1 (A. N. Verentchov, M. L. Vestal), US2002/0,117,616 A1 (M. L. Vestal). The first stage of the time-of-flight mass spectrometer demonstrates only moderate selectivity because the fragmentation always causes a slight smearing of the flight times of the ions.
The second method utilizes time-of-flight mass spectrometers with orthogonal injection (OTOF) of a continuous current of ions into an ion pulser, in which the ions experience a frequently-repeated sharply-pulsed orthogonal deflection into the flight path of the time-of-flight mass spectrometer. For tandem mass spectrometry, the parent ions are selected in suitable mass filters and fragmented in collision cells before being injected into the time-of-flight mass spectrometer. As a rule, they are selected in an RF quadrupole mass filter (Q), resulting in good selectivity, while fragmentation takes place in a further RF quadrupole collision cell. These instruments are abbreviated to QqOTOF, the lower case q indicating that what we have here is a collision cell and not a mass separating function. Methods and instruments of this type are described in detail in the patent specifications and patent publications EP 0 898 297 A2 (U.S. Pat. No. 6,107,623, R. H. Bateman, J. B. Hoyes), EP 1 220 290 A2 (R. H. Bateman, J. B. Hoyes), U.S. Pat. No. 6,285,027 B1 (I. Chernushevich, B. Thomson), WO 02/48 699 A2 (B. Thomson). The fragmentation occurs in the collision cells as a result of a large number of collisions at low collision energy, energy being absorbed by the complex internal oscillation system of the molecule until a statistical accumulation of the energy at a weak bond leads to a fragmentation of the molecule.
The fragmentation in a quadrupole collision cell, which is used in the second method using an OTOF, functions extremely well for doubly-charged ions, as are obtained in electrospray ion sources, for example. For matrix-assisted laser desorption (MALDI) producing practically only singly-charged ions, in contrast, the fragmentation in the collision cell is poor, since here only the bonds of the lowest possible binding energy break. If other bonds have a binding energy only higher by a few tenths of an electron-volt, so few of them break that the resulting fragment ions are no longer visible in the measured spectrum. The second method utilizing the OTOF, which has great advantages with respect to mass precision and selectivity, cannot therefore be used for ions from the MALDI process.
The high-energy collision process of the first method, on the other hand, achieves much better fragmentation of the singly-charged ions from the MALDI process. The fragmentation here occurs spontaneously at the first collision because sufficient energy is transferred. For peptides, the splitting take place statistically in the vicinity of the collision location, affecting all bonds between the amino acids. It is not only the bonds along the chain of the amino acids which are split, however. Side groups also split off, with a subsequent fragmentation of the chain by rearrangement processes.